One of the most compositionally diverse structures in a eukaryotic cell is a chromosome. A multitude of macromolecular protein interactions must properly occur on chromatin to drive functional aspects of chromosome biology like gene transcription, DNA replication, recombination, repair, and sister chromatid segregation. Importantly, unwanted alterations in PTM states and defects in the enzymatic machineries that regulate chromatin metabolism are linked to a wide variety of illnesses and diseases that are epigenetic in scope, including cancer. However, analyzing how protein assemblies interact in vivo with chromatin to direct these activities remains a significant challenge due to the temporal and dynamic nature of their associations. The in vivo analysis of macromolecular protein interactions in the context of chromatin has been a primary collaborative focus of the Taverna and Tackett laboratories for the last 6 years. Together, using the complementary skill sets of our groups, we have developed novel tools to unambiguously identify protein- protein interactions of macromolecular complexes on chromatin. Most importantly, our combined efforts recently culminated in the development of a technique termed Chromatin Affinity Purification with Mass Spectrometry or ChAP-MS. ChAP-MS provides for the enrichment of a unique 1 kb section of a chromosome for site-specific identification of macromolecular protein interactions and associated histone posttranslational modifications. The establishment of ChAP-MS in human cells will permit unprecedented insight into mammalian transcription regulation as well as epigenetic disregulation in human disease, and potentially mechanisms of action for therapeutics. In this proposal, we hypothesize that ChAP-MS can be adapted to human cell culture and tissues to provide a novel tool for the analysis of in vivo macromolecular protein interactions. Our short-term goal is to apply ChAP-MS to human cell lines and tissues, while our long-term goal is to move development into animal models like mice and to use ChAP-MS to profile across an entire chromosome. To test our hypothesis and work towards our short term goal, we will pursue the following three Aims: (1) Develop and Apply ChAP-MS for the analysis of macromolecular chromatin composition at actively transcribing genes in mammalian cell lines, (2) Develop an antisense enrichment procedure for ChAP-MS and (3) Application of Multiplex ChAP-MS in mammalian cells and tissues.